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Atypical cerebral listeriosis associated with Listera innocua in a beef bull. / Rocha P. R. D.; Dalmasso A.; Grattarola C. ;
Casalone C.; Del Piero F.; Bottero M.T.; Capucchio M.T... - In: RESEARCH IN VETERINARY SCIENCE. - ISSN
0034-5288. - STAMPA. - 94(1)(2013), pp. 111-114.
Original Citation:
Atypical cerebral listeriosis associated with Listera innocua in a beef bull.
Published version:
DOI:10.1016/j.rvsc.2012.07.017
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Atypical cerebral listeriosis associated with Listeria innocua in a beef bull
Paulo Ricardo Dell'Armelina Rocha
a*, Alessandra Dalmasso
a, Carla Grattarola
b, Cristina Casalone
b,
Fabio Del Piero
c, Maria Teresa Bottero
a, Maria Teresa Capucchio
aa Department of Animal Pathology, University of Turin, Italy
bIstituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta, Turin, Italy
c School of Veterinary Medicine, Department of Pathobiology, New Bolton Center, University of Pennsylvania, PA, United States
A B S T R A C T Keywords: Cattle Cerebral listeriosis Listeria innocua Meningoencephalitis
Natural infections of cattle associated with Listeria innocua have not been reported. This report describes the first case of cerebral listeriosis in a bull due to Listeria innocua. The animal presented neurological signs characterized by weakness, incoordination and recumbency. Histopathologic evaluation of brain tissue revealed multifocal microabscesses, perivascular lymphocytic cuffing, vasculitis, oedema and haemorrhages. All lesions extended from the medulla oblongata to the basal nuclei/parietal cortex area. Indirect immunohistochemistry labelled for Listeria sp. in the brain tissue, but not for Listeria
monocytog-enes, neurotropic Flaviviruses, BVDV, bovine Herpesvirus 1, Chlamydophila spp. and Histophilus somni. PCR
was negative for ovine herpesvirus. L. innocua was isolated from brainstem and identified by biochemical tests (Camp and beta-hemolysis negative). Subsequently, the species was confirmed by a duplex PCR and minisequencing assays. L. innocua should be histologically considered as a differential diagnosis of throm-botic meningoencephalitis, malignant catarrhal fever and cerebral listeriosis due to L. monocytogenes in cattle.
1. Introduction
Listeria spp. are Gram-positive facultative intracellular bacteria
ubiquitously distributed in the environment capable of growing at
a wide range of pHs and temperatures (Vazquez-Boland et al.,
2001). Listeria species are divided in two closely related lineages:
Listeria monocytogenes and Listeria innocua form one group, while
the second includes L welshimeri, L. ivanovii, and L seeligeri (Hain et al., 2006). Recently, two phylogenetically distant species L mar-thii (Graves et al., 2010) and L rocourtiae (Leclercq et al., 2010) were also described. Almost all cases of human listeriosis are due to L
monocytogenes; very rare infections due to Listeria ivanovii and Listeria seeligeri have been described (McLauchlin and Martin,
2008). L monocytogenes is also the major pathogen for other
ani-mals, although approximately 10% of septicaemia in sheep has been reported as due to L. ivanovii (McLauchlin and Martin, 2008). Wide-spread in the environment and in food, L innocua is generally con-sidered nonpathogenic (Vazquez-Boland et al., 2001). On the other hand, L. innocua has been associated with a human case of fatal sepsis and identified via blood culture and PCR assay (Perrin et al., 2003) and with a ewe that presented meningoencephalitis by brain
bacterial culture and PCR assay (Walker et al., 1994). There are no detailed gross, histopathologic and molecular descriptions of fatal L
innocua infection in cattle. The authors describe the neuropathology
of one case of Listeria innocua meningoencephalitis in a bull in Northwestern Italy.
2. Materials and methods
An 18 months old Blonde Aquitaine bull presented neurological signs characterized by weakness, incoordination and recumbency. The animal died after 1 day and was referred to the Department of Animal Pathology of Turin University for a complete necropsy. Dur-ing the post mortem, collected tissue samples were fixed in 10% neutral buffered formalin. Obex, pons, mesencephalon, cerebellum, hippocampus, thalamus, basal nuclei area, occipital, parietal and frontal cortex were transversally cut, routinely processed and embedded in paraffin. Five lm serial tissue sections were prepared for histopathologic and immunohistochemical examinations. Deparaffinized sections for histopathology were stained with hae-matoxylin and eosin (H&E).
Briefly, indirect immunohistochemistry was performed using primary antibodies against Listeria sp. (code 43251; Virostat, Port-land, USA), L monocytogenes (code 223021; Becton Company, Sparks, USA), Arboflaviviruses (Bioreliance, Rockville, MD, USA), Bovine Viral Diarrhea Virus (BVDV) (provided by Dr. E. J. Dubovi, College of Veterinary Medicine, Cornell University, Ithaca), bovine
Herpesvirus 1 (VMRD, Pullman, USA), Chlamydophila spp. (Chem-icon, USA), and Histophilus somni (provided by Dr. J. Lopez, Univers-idad Nacional Autonoma de Mexico, Mexico City). For antigen retrieval, all but one brain tissues were incubated in Proteinase K; citrate buffer (pH 6.0) was used for slides incubated with primary Listeria sp. antibody. The secondary antibodies were: immun-operoxidase staining using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, USA) for Listeria sp. slides; DAKO LSAB2-HRP (DAKO, Carpinteria, USA) secondary kit detection system (biotin labelled goat anti-rabbit + goat anti-mouse) for L. monocytogenes, bovine herpesvirus 1 and H. somni slides; and DAKO EnVision™+/HRP kit (DAKO, Carpinteria, USA, code K5361) for Arboflaviviruses, BVDV and Chlamydophila spp. Subsequently,
the 3,30-diaminobenzidine chromogen (DAB) was incubated for 1
min at room temperature. Finally, the sections were counterstained using Mayer's haematoxylin. Malignant catarrhal fever (MCF) ovine Herpesvirus polymerase chain reaction (PCR) was performed from paraffin embedded tissues.
A fresh sample of the malacic area in the brainstem (Fig. 1a and b) was submitted for bacterial isolation in Blood and MacConkey Agar at 37 °C for 48 h. Subsequently, the same samples were placed in liquid media (Fraser broth and Demi-Fraser broth) and incubated at 37 and 4 °C for 48 h. Cultures were then streaked onto Listeria Oxford Agar (Microbiol, Italy) and incubated at 37 °C for 48 h. To identify the isolate, a miniaturized system was used (colorimet-ric Vitek 2 identification system, bioMerieux Inc, Bagno a Ripoli, Italy), as well as further CAMP and betahaemolysis tests.
For the confirmation of the species of the isolate, biomolecular assays (Dalmasso et al., 2010) were also performed from the broth cultures and from a frozen sample of the brainstem. DNA was ex-tracted by boiling. A duplex PCR characterized by a specific frag-ment on phosphatidylinositol-specific phospholipase C (plcA) gene for L. monocytogenes (632 bp long) and by a common fragment of 16S rRNA gene for all Listeria spp. (437 bp long) was performed. When both bands were detected in the agarose gels, L.
monocytogenes was immediately identified. When only the
437-bp band was present, it was possible to identify the Listeria genus but not to discriminate between the other Listeria species (L
innoc-ua, L. welshimeri, L. seeligeri, L. ivanovii, and L grayi). In this
case, the amplified product (437-bp band) was purified and submitted to a
minisequencing reaction able to detect diagnostic sites that are able to identify five Listeria (already mentioned above; according to Dalmasso et al., 2010).
3. Results
At necropsy no significant macroscopic findings were noted in organs or tissues other than the brain. Transversal sections of the brain revealed dark brown areas interpreted as malacic foci involv-ing the obex, pons, mesencephalon, thalamus and basal nuclei. Encephalomalacia was prominent in the obex and the right part of the pons. These lesions continued rostrally involving the right portion of the brain to the basal nuclei area as well as the adjacent parietal cerebral cortex (Fig. 1a-d).
Microscopically, lesions were moderate to severe, including multifocal microabscesses, vasculitis, perivascular lymphocytic
cuffing, oedema and haemorrhages (Fig. 2a-d). Microabscesses
were characterized by multifocal randomly distributed aggregates of neutrophils, small to coalescing foci of liquefactive necrosis and oedema. In the areas adjacent to the microabscesses there was multifocal gliosis, diffuse infiltration of neutrophils and microglial cells as well as neuronal necrosis. The microabscesses particularly involved the pons, cerebellum, thalamus and adjacent cerebral cor-tex. Necrotizing vasculitis with frequently associated perivascular haemorrhage was also observed within the brainstem, cerebellum, and white matter of the cerebral cortex. Perivascular cuffing was rarely observed in the obex, pons and midbrain and was composed of one to two layers of lymphocytes and occasional macrophages. Haemorrhages were observed in all areas of the brain, but were more severe in the cerebellum, thalamus and basal nuclei, where coalescing microabscesses were mostly observed. Multifocal mod-erate mononuclear leptomeningitis adjacent to microabscesses was also observed in the brainstem and cerebellum. Vagus, trigem-inal and facial brainstem nuclei were affected by non-coalescing
microabscesses and focal perivascular cuffing.
Immunohistochem-istry for Listeria sp. allowed for the identification of the bacteria within the microabscesses, with the highest intensity noted in the cerebellum, thalamus (Fig. 2d) and mesencephalon. Listeria antigen was observed in the cytoplasm of mononuclear and
Fig. 2. (a) Brain, thalamus, coalescing microabscesses with malacic area (arrows) and vasculitis (asterisks) (HE). (b) Brain, thalamus, vasculitis (arrow) surrounded by multifocal to coalescing microabscesses (HE). (c) Brain, obex, microabscess affecting the vagus nuclei (HE). (d) Brain, cerebellum, brown areas correspond to immunopositivity for Listeria sp. antibody in a malacic area (DAB chromogen, counterstained with haematoxylin).
polymorphonuclear cells (resembling gitter cells and neutrophils, respectively) of microabscesses and rarely within the lumen of blood vessels affected by vasculitis. L. monocytogenes, neurotropic Flaviviruses, BVDV, bovine Herpesvirus 1, Chlamydophila spp. and
H. somni were not present. MCF ovine herpesvirus was not detected
by PCR.
L innocua was isolated only from the brainstem and the identity
of the unusual isolate was confirmed by the Vitek 2 identification system and by Camp and betahaemolysis negative results. Duplex PCR (Fig. 3) of both the frozen brainstem sample and the bacteriol-ogy culture allowed the amplification of only a 16S rRNA gene 437-bp fragment in the isolate, indicating that this isolate was of
Listeria genus, but not L monocytogenes. L. monocytogenes, L. innocua
and other Listeria spp. present single nucleotide polymorphism in
Fig. 3. Duplex PCR. M: marker, 100bp; PC1: positive control (L monocytogenes); PC2: positive control (L innocua); S: isolate of the present report, which amplified only a 467 bp fragment, as did PC2; NC: negative control (ultrapure water).
nucleotides 93, 178, 226, and 270 relative to L innocua 16S rRNA gene sequence (GeneBank Accession No. X56152). For bothL.
mon-ocytogenes and L innocua, nucleotides corresponding to these
posi-tions are A,G,G,T, respectively. These polymorphisms were detected with a minisequencing assay (Dalmasso et al., 2010). To-gether, minisequencing analysis result (data not shown) with the lack of amplification of the L. monocytogenes-specific fragment in the duplex PCR (Fig. 3) led us to conclude that the identified species was indeed L innocua.
4. Discussion
Bovine cerebral listeriosis is classically caused by L
monocytog-enes, while other species of Listeria have been traditionally
classi-fied as non-pathogenic (Low and Donachie, 1997). The
microscopic lesions of the present case were similar to acute cere-bral listeriosis due to L monocytogenes in ruminants (Oevermann et al., 2010). However, vasculitis in this instance was particularly severe and was often associated with large areas of necrosis and haemorrhage. Moreover, the microabscesses and areas of enceph-alomalacia were more evident and extensive than those described in the literature of typical cerebral listeriosis lesions in
cattle (Ladds et al., 1974). Walker et al. (1994) described
meningoencephalitis due to L innocua in a ewe in which histopathologic examination noted moderate mononuclear perivascular cuffing and neutrophilic margination. Vasculitis was also reported - as in the present case - but was restricted to the midbrain. Furthermore, lesions (Walker et al., 1994) were limited in distribution and severity in comparison with the present case and with cases of classical encephalitic listeriosis of sheep (Ladds et al., 1974), and microabscesses were not detected.
Other diseases, such as thrombotic meningoencephalitis (TBME) - caused by H. somni - or MCF can also cause vascular le-sions similar to the present case in the brain of cattle (Summers et
al., 1995; Maxie and Youssef, 2007). However, TBME generally
causes severe vasculitis and thrombosis, with or without
infarction; there is a predilection for the thalamus and the junction
of the white and gray matter of the cerebral cortex (Maxie and
Youssef, 2007).
MCF infection is epidemiologically associated with cattle having
contact with sheep or wild ruminants (Russell et al., 2009). There
was no history of contact with these animals in this case. Addition-ally, MCF causes lymphocytic and histiocytic vasculitis with severe fibrinoid necrosis involving the tunica media and adventitia of the arteries and arterioles. Additionally, MCF in ruminants often causes erosive or ulcerative lesions in the gastrointestinal and respiratory tracts, as well as lymphoid hyperplasia (Summers et al., 1995), as opposed to the neutrophilic pathology associated with listeriosis.
Although Rocourt and Seeliger (1985) previously reported isola-tion of L innocua from the brain of cattle experimentally infected and concluded that it was non-pathogenic, the present paper con-stitutes the first description of natural infection, brain lesions, bac-teriology, immunohistochemic positivity and molecular biology confirmation of L innocua infection in the brain of a bovine animal. Haemolysis is considered an important test to distinguish between
L. monocytogenes and L. innocua (Gorski, 2008). However, misinter-pretation from biochemical profiles of Listeria genus has been
re-ported. Indeed, Johnson et al. (2004) reported that L. innocua may
ocasionally present b-haemolytic activity, which is a virulence gene-related feature usually associated with L monocytogenes. Further molecular characterization of this isolate revealed the
presence of internalin A virulence gene (Volokhov et al., 2007).
Non-haemolytic strains of L. monocytogenes have also been de-scribed (Allerberger et al., 1997). Therefore, the species affirmation
by Rocourt and Seeliger (1985), based only from biochemical
characteristics, is uncertain.
Co-infection with L monocytogenes or other biological agents seems unlikely in this instance, since L. innocua was isolated from the brainstem. Finally, immunohistochemistry and PCR assays for other previously described pathogens were negative, ruling out the potential for co-infection. Although it was not possible to eval-uate the immune status of the present bull, the authors suggest that this atypical infection may be caused by a combination of immunosuppression and a mutated bacterial strain. In fact, natural heterogeneity in avirulent Listeria species, reflected in its ability to acquire or retain virulence-associated genes, may permit some L.
innocua to be causative agents of disease, especially in
immuno-compromised mammals (Volokhov et al., 2007).
To the best of the author's knowledge, this is the first descrip-tion of fatal meningoencephalitis caused by L innocua in a bull. Therefore, the authors recommend to increase the use of microbi-ological and biomolecular techniques in all suspected cases of lis-teriosis additionally to the traditional histopathology and immunohistochemistry for a correct aetiological diagnosis. Fur-thermore, L innocua should be histologically considered as a differ-ential diagnosis of TBME, MCF and cerebral listeriosis due to L.
monocytogenes in cattle.
Conflict of interest statement
The authors declared that they had no conflicts of interest with respect to their authorship or the publication of this article.
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